An expression system of rat calmodulin using T7 phage promoter in Escherichia coli.
An efficient expression system of rat calmodulin in Escherichia coli is presented. To express rat calmodulin cDNA, we employed a pET expression vector which contains the T7 phage promoter and terminator. After transformation of E. coli BL21(DE3) strain which carries T7 phage RNA polymerase inducible with isopropyl-beta-D-thiogalactopyranoside, induction of the expression, and chromatography of soluble proteins on a phenyl-Sepharose column, about 250 mg of recombinant rat calmodulin was obtained from 1 liter of E. coli culture. The recombinant calmodulin lacked the N-terminal methionine, and posttranslational modifications such as Nalpha-acetylation and methylation. This system facilitates the large amount preparation of calmodulin and the mutant proteins required for the structural analysis by NMR spectrometry and/or X-ray crystallography.
Hayashi
N
N
Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi, 470-11, Japan. nhayashi@fujita-hu.ac.jp
Matsubara
M
M
Takasaki
A
A
Titani
K
K
Taniguchi
H
H
D000818:Animals; D017123:Bacteriophage T7 / Q000235:genetics*; D001483:Base Sequence; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D009682:Magnetic Resonance Spectroscopy; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D002147:Calmodulin; D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D009219:Myosin-Light-Chain Kinase